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PlasmidDNAPreparationforRecombination
LinChengyuBio042010030007;Cooperator:YuanXiaowen
ExperimentDate:2012-03-15;SubmitDate:2012-03-18
1Introduction
Plasmidisasmall,independentlyreplicatingandcytoplasmicDNAmoleculethatcan
-strandedandusually
circularDNA,whosesizerangefrom1kbto200kb.
Figure1Sketchofplasmidnucleoidandcell
Becauseofitsproperty,plasmidisoftenusedascloningvectorcarryingforeigngenes
,several
preparativeprocessesshallbedone,includingplasmidDNAextraction,restriction
enzymedigestionandagarosegelelectrophoresis.
(1)Learnthecharacteristicsofplasmid.
(2)PurifytheplasmidDNAbyalkalinelysismethod.
(3)MeasuretheDNAconcentrationbyspectrophotometer.
(4)Learnthecharacteristicsofrestrictionendonuclease,anduseEcoRIandXhoIto
performrestrictionenzymedigestion.
(5)PerformagarosegelelectrophoresistoseparateDNAs,andcomparethedifference
amongoriginalplasmids,singledigestion,anddoubledigestion.
(1)Somespecialelements,includingheat,extremepH,organicsolutioncan
denatureDNAandwillbringDNAintosediment;
(2)Whendenaturingconditionsareeliminated,DNAwillberenatured,andits
speedwilldependonthesizeandratiooverprotein;
(3)Theisolationkithasspecialmaterialonthecolumnandcanspecifically
,plasmidscanbepurified;
(4)Double-
ExperimentReportonMolecularBiology,2012
concentrationcanbemeasuredbyspectrophotometer.
(1)TypeIIrestrictionendonucleasecanrecognizespecificnucleicacid
sequenceandincisedoublestrainDNAinspecificsitesinthissequence.
Figure2illustratethespecificsequenceEcoRIrecognizeanditscutting
site;
Figure2CuttingsiteofEcoRI
(2)Mostplasmidshavemultiplecloningsites(MSC)whichisartificially
designforgeneticengineering(SeeFigure3).Restrictionendonucleasecan
digestinthespecificsite,producestickyendsforrecombination.
Figure3Plasmidprofile(pCMV-Myc)
Inordertoconfirmthatthepreparationaboveissuccessful,agarosegel
electrophoresisisessential.
Agarosegelhassmallporesinit,whosesizedependsonagarose
,negativelycharged
ExperimentReportonMolecularBiology,2012
andDNAconformationwillbethemajorfactorsthatdeterminethemobility
,DNAofdifferentsizesandconformationscanbe
,thesizeofsamplecanbeknownaswell.(See
Figure4)
Figure4Agarosegelelectrophoresis:
(1)plasmids(2)EcoRIdigestion(3)doubledigestion1
2ExperimentOperation
-Myc-T10(SIPAR),
EcoRIandXhoIrestrictionendonuclease(inglycerol,fordetailsseeTable1),
Table1RecognitionSequenceandcuttingsiteofEcoRIandXhoI2
EnzymeRecognitionSequence
EcoRIG’AATT^C
XhoIC’TCGA^G
NEB1kbDNALadder(fordetailsseeFigure5);
Figure5NEB1kbDNALadder3
ExperimentReportonMolecularBiology,2012
Table2Solutions
Solution/BufferCompositionandConcentration
50mMglucose
10mMEDTA
SolutionI(GETbuffer)
25mMTris-HCl()
AddDNase-freeRNasebeforeuse
SolutionII(fresh)
1%SDS
60mL5MKAc
2
BindingbufferGuanidine()
80mMKAc
-HCl()
Washingbuffer
40µMEDTA
add3foldsvolumeofabsoluteethanolbeforeuse
ElutionbufferddHO
2
10×Hbuffer
1×TAEbuffer
%Bromophenolblue
3×Loadingbuffer
30%sucrose
EB10mg/mlEthidiumbromide
(1)Pipettes;
(2)Eppendorftube;
(3)Biodevplasmidisolationkit;
(4)Centrifuge;
(5)Spectrophotometer;
(6)Electrophoresisapparatus;
(7)UVgelimagingsystem.
(1),
centrifugeat12,000rpmfor2min,
,andrepeat;
(2)Add100µlSolutionI(containsDNase-freeRNase),resuspendpellet
.;
Notes:,
avoidingharshenvironment.
(3)Add150µlSolutionII,mixbyinvertingthetube4-
;
ExperimentReportonMolecularBiology,2012
Notes:SolutionIIcontainsNaOHandSDS,whichprovideastrongly
,
solubilityinwaterislowered.
(4)Add150µlSolutionIII,mixbyinvertingthetubegentlyseveraltimes.
5mintogetgenomicDNAprecipitatecompletely,andcentrifugeat12,000
rpmfor8min;
Notes:SolutionIIIcanneutralizethedenaturingenvironmentcausedby
SolutionII,
willbereplacedwithpotassiumioninSolutionIII,andbecomesPDS
(PotassiumDodecylSulfate),,
combinedwithproteinandgenomicDNA,willcoprecipitateintopellet.
(Fordetails,seediscussionsession.)Usingcentrifugecanremove
genomicDNA;
(5)Add420µlBindingbuffertothemini-spincolumntoactivateitsabilityto
bindDNAmolecules;
(6)TransferthesupernatantfromStep4tothemini-spincolumn,mixwith
bindingbufferbypipette,placethemini-spincolumninthecollectiontube,
andcentrifugeat12,
collectiontube;
Notes:Atthistime,plasmidsspecificallybindtothematerialinthe
mini-spincolumn;
(7)Add700µlWashbuffer(contains3foldsvolumeofabsoluteethanol)to
themini-spincolumn,andcentrifugeat12,
µl;
Notes:Washbuffercanwashoffthenon-DNAsubstancethat
non-specificallybindstothemini-
becauseplasmidswillbewashedoffwithitsabsence.
(8)Centrifugethemini-spincolumnaswellascollectiontubeat12,000rpm
for2mintogetridofethanol;
Notes:
ethanolfromthecolumn.
(9)ReplacethecollectiontubewithanewEppendorftube,add50µlddHOto
2
themini-spincolumn,.,andcentrifugeat12,000rpm
for1min;
(10)LabeltheEppendorftubecontainingplasmidsatinitialconcentrationwith
class,name,grouponit;
(11)Pipette96µlddHOintoanewEppendorftube,andadd4µlofplasmid
2
.
Notes:[dsDNA]=50*(OD–OD)*dilutionfactor.
260310
(1),2,3;
ExperimentReportonMolecularBiology,2012
(2)Addsubstrate,solutions,andenzymesasshowninTable3;
Table3Schemeofrestrictionendonucleasedigestion
Plasmid
10×HEcoRI/XhoI/
Number(/µlTotal/µl
buffer/µlµlµl2
µl)/µl
(3)Incubate3tubesat37℃for30min;
(1)Duringenzymedigestion,placeacleanelectrophoresismoldona
horizontalsurface,carefullyinsertacomb(15µlslots)intothemold;
(2)Pourtheagarosegelsolution()into
thantheblacklineonthesideface;
(3)Waitforapproximately30minforsolidification,whenenzymaticreactions
completeaswell;
(4)Add10µl3×LoadingbuffertoeachEppendorftube,andmixwithpipette;
(5)Carefullyremovethecomb,placethemoldintheelectrophoresischamber,
andadd1×TAEbufferinordertocoverthegeltoadepthof
approximately1mm;
(6)Load15µlfromeachEppendorftubeintotheslotsofthegel:oneslotfor
µlof1kbDNAladderintotheslotbesidethesample;
Notes:
startslateaftersamplesloading,samplesmightdiffuseinthegelandcause
thebandgetintheendnotparallelwiththeslot.
(7)Startelectrophoresisimmediatelysamplesloadingat100V,andstopwhen
Bromophenolblueis2/3gellengthfromthestartingline;
(8)PlacethegelintoEBworkingsolutionfor20min,andobserveunderUV
;
3Result&Discussion
Table4Plasmidconcentrationandpurity
ConcentrationODODOD/OD
260280260280
/µ
/µl,/
260
~
280
fromtheEppendorftubetothemini-spincolumn,thepelletwaslooselyattached,
causingsomeproteinsolute,andmakingtheextractionnotpureenough.
Q1:Duringplasmidextraction,whenSolutionIIwasadded,whycan’tincubatelonger
ExperimentReportonMolecularBiology,2012
than2min?
A1:NaOHcanproduceastrongalkalineenvironmenttolysecells,butgenomicDNA
willalsodegradedslowlyunderalkalinesituation.
Q2:WhenSolutionIIIisadded,?
A2:Inordertostudythenatureofthepellet,Idesignedanexperimentandperformitin
thelab.
Table5ExperimentdesignedtostudythepelletafterSolutionIIIadded
+SolutionI+Whitepellet
SolutionII+SolutionIII
(negativecontrol)
+SolutionI+Littleamountofwhitepellet
SolutionIIwithoutSDS+
SolutionIII
+SolutionI+Nopellet
SolutionIIwithoutNaOH+
SolutionIII
41%SDS+2MHAcNopellet
51%SDS+5MNaClSDSprecipitate
61%SDS+3MKClLargeamountofPDSprecipitate
Fromtheresult,wecanknowthatNaOHismainlyusedforlyse,anddenaturedDNAis
stillsoluble!
acidicandhighsaltconcentrationenvironment.
,as
demonstratedamongexperiment4,5,,PDSprecipitateinalarge
amount,
DNAmoleculesarebindwithproteins,andarebroughtintopelletinthesametime.
TheresultisshowninFigure6.
Figure6PlasmidDNAgelelectrophoresis:
LaneI–Plasmidwithouttreatment;
LaneII–EcoRIsingledigestion;
LaneIII–EcoRIandXhoIdoubledigestion;
LaneM–1kbDNALadder
ExperimentReportonMolecularBiology,2012
AsshowninFigure6,LaneIII,whichcontainsplasmidstreatedbydoubledigestion,
,;LaneII,which
containsplasmidstreatedbyEcoRIsingledigestion,,which
isaboutthesamelengthasthesumoftwocompositionfromLaneIII.
ThisphenomenonsuggeststhatinpCMV-Myc-T10,thenumberofEcoRIrecognition
,bothenzymehaveand
onlyhaveonerecognitionsite,whichconfirmourconjecture.
AsforLaneI,
,wefailedtoputthepipette
nearthebottomoftheslot,butthemiddleofit,causingalotofDNAmolecules
overflowfromtheslot.
4Conclusion
/µ
,has1EcoRIand1XhoIrestrictionenzyme
recognitionsite.
5Reference
【1】Resultsofstudentexperiment,2004-03-15
【2】
【3】
6Appendix
PlasmidDNAextractionprocedureincludesdenaturingandrenaturing,inorderto
removegenomicDNA;genomicDNAextractiontendstofocusmoreonprotein
removal,andwillinevitablymixwithplasmidDNAifwithoutfurtherpurification.
ThegenesequencecomesfromNCBI,whichcodingforPDCD11(ProgrammedCell
Death),alsonamedasALG-4(Apoptosis-LinkedGene).Itiscomposedof6000bp.
UsingBioedittoanalyze:
Use“Sequence->Selectpositions”toselectthewholesequence;
Use“Sequence->Nucleicacid->Restrictionmap”:
Figure7-1
ExperimentReportonMolecularBiology,2012
Click“GenerateMap”,hereshowsanalysisfrom881to960bp:
Figure7-2
Andthoseenzymesthathavenorecognitionsitesarelistedasfollows:
Figure7-3